Method for plant transformation based on thje pollination-fecundation pathway and the products thereof

ABSTRACT

A genotype-independent method for efficiently carrying out pollen-mediated gene transformation of cereals uses pollen which is pretreated with silicon carbide, and transforming DNA coding for specific traits, whereby the method can produce transformed plants exhibiting useful traits, with high efficiency and reproducibility.

BACKGROUND OF THE INVENTION

The present invention relates to methods for plant genetictransformation and for products thereof. More specifically, the presentinvention relates to the genetic transformation of any plant specieswith sexual reproduction based on a pollination-fecundation process.According to the present invention, pollen grains are pre-treated withsilicon carbide fibers and the transforming DNA. The present inventionalso involves pollinating recipient plants with pollen grains carryingthe transforming DNA.

Advances in molecular biology have dramatically expanded man's abilityto manipulate the germplasm of animals and plants. Genes controllingspecific phenotypes, for example specific polypeptides that lendantibiotic or herbicide resistance, have been located within a certaingermplasm and isolated from it. Even more important has been the abilityto take the genes which have been isolated from one organism and tointroduce them into another organism. This transformation may beaccomplished even where the recipient organism is from a differentphylum, genus or species from that which donated the gene (heterologoustransformation).

Genetic engineering of plants, which entails the isolation andmanipulation of genetic material (usually in the form of DNA or RNA) andthe subsequent introduction of that genetic material into a plant orplant cells, offers considerable promise to modem agriculture and plantbreeding. Increased crop food values, higher yields, feed value, reducedproduction costs, pest resistance, stress tolerance, drought resistance,the production of pharmaceuticals, chemicals and biological molecules aswell as other beneficial traits are all potentially achievable throughgenetic engineering techniques.

Once a gene has been identified, cloned, and engineered, it is stillnecessary to introduce it into a plant of interest in such a manner thatthe resulting plant is both fertile and capable of passing the gene onto its progeny.

Developments in agrobiotechnology have resulted in a tremendousexpansion of the capabilities for the genetic engineering of plants.Many transgenic dicotyledonous plant species have been obtained.However, many species of plants, especially those belonging to theMonocotyledonae and particularly the Gramineae, including economicallyimportant species such as corn, wheat and rice, have proved to be veryrecalcitrant to stable genetic transformation. Difficulties have beenencountered in achieving both: a) integrative transformation of monocotplant cells with DNA (i.e., the stable insertion of DNA into the nucleargenome of the monocot plant cells) and b) regeneration from transformedcells of phenotypically normal monocot plants, such as phenotypicallynormal, fertile adult monocot plants. It has been suggested that suchdifficulties have been predominantly due to the nonavailability ofmonocot cells that are competent with respect to: 1) DNA uptake, 2)integrative transformation with the taken-up DNA, and 3) regeneration ofphenotypically normal, monocot plants from the transformed cells(Potrykus (1990) Bio/Technology 9:53 5).

Thus, the introduction of exogenous DNA into monocotyledonous speciesand subsequent regeneration of transformed plants has proven much moredifficult than transformation and regeneration in dicotyledonous plants.Moreover, reports of methods for the transformation of monocotyledonssuch as maize, and subsequent production of fertile maize plants, havenot been forthcoming. Consequently, success has not been achieved inthis area and commercial implementation of transformation by productionof fertile transgenic plants has not been achieved. Thus there is aparticularly great need for methods for improving geneticcharacteristics. Problems in the development of genetically transformedmonocotyledonous species have arisen in many general areas. For example,there is generally a lack of methods which allow one to introducenucleic acids into cells and yet permit efficient cell culture andeventual regeneration of fertile plants.

Genetic engineering techniques have been successfully appliedprincipally in dicotyledonous species. The uptake of new DNA byrecipient plant cells has been accomplished by various means, includingAgrobacterium infection (Nester, E. W., et al, (1984). Ann. Rev. PlantPhysiol 35:387-413), polyethylene glycol (PEG) mediated DNA uptake (LorzH., Baker B., Schell J. (1985). Mol Gen Genet 199:178-182.),electroporation of protoplasts (Fromm M. E., Taylor L. P., Walbot V.(1986). Nature 312:791-793.) and microprojectile bombardment (Klein T.M., Kornstein L., Sanford J. C., Fromm M. E. (1987). Nature 327:70-73.).

The Agrobacterium transformation system is among the recombinant DNAtechnologies for genetic manipulation of plant genotypes. Virulentstrains of the soil bacterium Agrobacterium tunlefaciens are known toinfect dicotyledonous plants and to elicit a neoplastic, response inthese plants. The tumor-inducing agent in the bacterium is a plasmidthat functions by transferring some of its DNA into its host plant'scells where it is integrated into the chromosomes of the host plant'scells. This plasmid is called the Ti plasmid, and the virulence of thevarious strains of A. lumefaciens is determined in part by the virregion of the Ti plasmid which is responsible for mobilization andtransfer of the T-DNA (Schell, J., Science, 237:1176-1183 (1987)). TheT-DNA section is delimited by two 23-base-pair repeats designated asright border and left border, respectively. Any genetic informationplaced between these two border sequences may be mobilized and deliveredto a susceptible host. Once incorporated into a chromosome, the T-DNAgenes behave like normal dominant plant genes. They are stablymaintained, expressed and sexually transmitted by transformed plants,and they are inherited in normal Mendelian fashion.

There are two common ways to transform plant cells with Agrobacterium:co-cultivation of Agrobacterium with cultured isolated protoplasts, ortransformation of intact cells or tissues with Agrobacterium. The firstrequires an established culture system that allows for culturingprotoplasts and subsequent plant regeneration from cultured protoplasts.The second method requires (a) that the intact plant tissues, such ascotyledons, can be transformed by Agrobacterium and (b) that thetransformed cells or tissues can be induced to regenerate into wholeplants.

Agrobacterium-mediated transformation in dicotyledons facilitates thedelivery of larger pieces of heterologous nucleic acid as compared withother transformation methods such as particle bombardment,electroporation, polyethylene glycol-mediated transformation methods,and the like. In addition, Agrobaclerium-mediated transformation appearsto result in relatively few gene rearrangements and more typicallyresults in the integration of low numbers of gene copies into the plantchromosome.

However, the Agrobacterium transformation system, as stated, isrestricted to certain dicot crops. For the majority of monocots,especially cereals (graminae) and grasses, A.tumefaciens mediated genetransfer is not possible. Thus, the most important cultivated plants arenot accessible for effective gene transfer.

A second frequently used process for transformation of plants is DNAdirect delivery. One form of direct DNA delivery is direct gene transferinto protoplasts (using polyethylene glycol treatment and/orelectroporation). Protoplasts for use in such direct gene transfermethods have most often been obtained from embryogenic cell suspensioncultures (Lazzeri and Lorz (1988) Advances in Cell Culture, Vol. 6,Academic press, p. 291; Ozias-Akins and Lorz (1984) Trends inBiotechnology 2:119). However, the success of such methods has beenlimited due to the fact that regeneration of phenotypically normalplants from protoplasts has been difficult to achieve for mostgenotypes. For example, while regeneration of fertile corn plants fromprotoplasts has been reported, these reported methods have been limitedto the use of non-transformed protoplasts. Moreover, regeneration ofplants from protoplasts is a technique which carries its own set ofsignificant drawbacks. Even with vigorous attempts to achieve fertile,transformed maize plants, reports of success in this regard have notbeen forthcoming.

In yet another form of direct transformation, the genetic material istransferred using high velocity ballistic penetration by small particleswith the nucleic acid either within the matrix of small beads orparticles, or on the surface (Klein, et at, Nature, 327:70-73 (1987)).In this method, non-biological particles may be coated with nucleicacids and delivered into cells by a propelling force. Exemplaryparticles include those comprised of tungsten, gold, platinum, and thelike. The main advantage of particle bombardment over Agrobacterium isabsence of biological incompatibilities found when using this biologicalvector. In the plant kingdom, particle bombardment has shown goodutility for transformation of conifers, dicots and monocots. However,particle bombardment has certain drawbacks relating to cost, ease ofuse, accessibility and end product utility. Moreover, transgenic plantsobtained via Agrobacterium generally contain more predictable introducedDNA's while particle bombardment, as well as other direct DNA uptakemethods, give rise to more random and uncontrolled DNA integrationevents. Particle bombardment also often results in complex transgeneinsertion loci, which may cause gene silencing in some instances. Inaddition to their restrictive application in dicoytyledoneae andrelatively low transformation rates, these systems require theregeneration of entire plants from plant protoplasts.

Thus, great difficulties remain also in employing methods of direct DNAdelivery, due to the high dependence on the ability of the genotype toregenerate. As a consequence, in the few known examples of successfultransformation of maize the experimental material was based on the lineA188 which is easy to regenerate. Noteworthy, in all of the methodsbased on the multicellular target (embryos, leaf-discs or calli) is thefact that the resulting transformed tissue is mosaic, demanding furthersteps to obtain non-mosaic progeny. Most of these difficulties are dueto the use of long-term tissue culturing.

Another major problem in achieving successful monocot transformation hasresulted from the lack of efficient marker gene systems which have beenemployed to identify stably transformed cells. Marker gene systems arethose which allow the selection of, and/or screening for, expressionproducts of DNA. For use as assays for transformed cells, the selectableor screenable products should be those from genetic constructsintroduced into the recipient cells. Hence, such marker genes can beused to identify stable transformants.

Of the more commonly used marker gene systems are gene systems whichconfer resistance to arninoglycosides such as kanamycin. While kanamycinresistance has been used successfully in both rice (Yang et al, 1988)and corn protoplast systems (Rodes et al, 1988), it remains a verydifficult selective agent to use in monocots due to high endogenousresistance (Hauptmann, et al, 1988). Many monocot species, maize, inparticular, possess high endogenous levels of resistance toaminoglycosides. Consequently, this class of compounds cannot be usedreproducibly to distinguish transformed from non-transformed tissue. Newmethods for the reproducible selection of or screening for thetransformed plant cells are therefore needed. Accordingly, it is clearthat improved methods and/or approaches to the genetic transformation ofmonocotyledonous species would represent a great advance in the art.Furthermore, it would be of particular significance to provide novelapproaches to monocot transformation, such as transformation of maizecells, which would allow for the production of stably transformed,fertile corn plants and progeny into which desired exogenous genes havebeen introduced. The identification of marker gene systems applicable tomonocot systems such as maize would provide a useful means for applyingsuch techniques generally. The development of these and other techniquesfor the preparation of stable genetically transformed monocots such asmaize could potentially revolutionize approaches to monocot breeding.

In order to overcome the difficulties of genotype-dependenttransformation caused by low regeneration potential of cereals, manyefforts were put to develop an alternative, genotype-independenttransformation approach based on the pollination pathway (Ohta Y.,1986). In maize, high efficiency genetic transformation can be achievedby a mixture of pollen and exogenous DNA. (Luo Z X and Wu R., 1988,Proc. Natl. Acad. Sci. USA 83:715-719). Maize, often referred to as cornin the United Stated, can be bred by both self-pollination andcross-pollination techniques. Maize has separate male and female flowerson the same plant, located on the tassel and the ear, respectively.Natural pollination occurs in maize when wind blows pollen from thetassels to the silks that protrude from the tops of the ears.Transformation of rice via the pollen-tube pathway has also beendemonstrated (Plant Molecular Biology Reporter 6:165-174). The majorpotential advantages of the pollen-tube pathway approach include: (a)genotype independence; (b) lack of mosaicism; (c) no need forcomplicated cell and tissue culture techniques.

Despite the keen interest in an effective transformation method havingsuch advantages, no serious results have been obtained with thisapproach, because of low reproducibility. Nevertheless, partial transferof alien genes into intact plants via pollination pathway has beenreported in maize, tomato and melon (Chesnokov, Yu. V., et al, 1992,USSR Patent No. 1708849; Bulletin of the USSR Patents, No. 4; ChesnokovYu. V. & Korol A. B. 1993; Genetika USSR, 29:1345-1355).

The procedures of genetic transformation based on thepollination-fecundation pathway include: (1) employment of a mixture(paste) of the pollen and transforming DNA; (ii) delivery of the alienDNA into the pollen tube, after pollination; and (iii) microparticlebombardment of microspores or pollen grains. The obstacles inapplication of the so-far developed versions of the pollination pathwayof genetic transformation include: (i) very low reproducibility; (ii)extremely poor applicability to maize due to the very long style of thisplant; and (iii) high cost (Potrykus, I. 1990. Gene transfer to cereals:an assessment. Bio/Technology 8:535-542). The present invention providesan alternative highly efficient method of plant genetic transformationand in particular of maize genetic transformation employing pollentreatment with silicon carbide fibers in the presence of foreign DNA.

Silicon carbide fiber technique has been used in plant genetictransformation procedures based on tissue culturing (Kaeppler, H. F.,Somers, D. A., Rifles, H. W. and Cockburn, A. F. 1992. Silicon carbidefiber-mediated stable transformation of plant cells. Theor. Appl. Genet.84:560-566). Such an approach, is restricted by low regenerationpotential of cereals in general, and maize in particular, limiting itsapplication to elite cultivars. Moreover, this method provides onlyabout 10% of the efficiency achieved by microparticle bombardment of theembryogenic tissues.

The present invention combines an improved process of pollinationpathway and silicon fiber treatment that permits solving the abovementioned problems by delivering the transforming DNA into pollen grainsand then, via the sperm, into the egg cells. This novel and non-obvioussolution allows to achieve high frequency of maize transformation, andin other crops as well. Beside high efficiency and low cost, its mostimportant advantages are high reproducibility, genotype independence,genetic stability of the transformants, and technical simplicity. Thesefeatures, taken together, comprise a novel combination which allows saidinvention to become a basis for large-scale genetic transformation,especially in maize, but in other crops as well. The uniqueness ofcombining the pollination pathway and the delivering of the transformingDNA into pollen grains by silicon carbide fibers is that the methodtakes advantages of the natural reproduction system resulting intransformed zygotes.

The advantages of the developed strategy include: (1) expensive andtime-consuming tissue culture techniques are not required, (2)genotype-independence, since the method does not require in vitroregeneration procedures, (3) elimination of plant sectoring (mosaicism),since the transformants result from zygotes, (4) no somaclonal variationand reduced fertility caused by prolonged tissue culturing, (5) the useof natural delivery system ensures high stability of the integrated DNA,(6) potential to transfer large fragments of alien DNA into the plantgenome; and (7) low cost, high frequency and reproducibility.

Another important advantage of the present method is the possibility ofusing it for plant transformation (primarily cereals) by large fragmentsof DNA, e.g. cloned in yeast artificial chromosomes. This allows anincrease in the efficiency of map-based cloning of genes of agronomicalimportance.

SUMMARY OF THE INVENTION

A method for plant transformation with resistant properties againstantibiotics, herbicides as well as enhanced anthocyanin is provided.

The present invention is directed to a method for genetic transformationof any plant species with sexual reproduction based on apollination-fecundation process, and its products thereof. According tothe present invention the recipient plants are pollinated by pollengrains carrying the transforming DNA wherein the pollen grains arepre-treated by silicon carbide fibers and the transforming DNA.Accordingly, the present invention provides an improved process whichcombines the pollination pathway and the delivery of the transformingDNA into pollen grains by silicon carbide fibers. The method also allowsthe possibility to conduct controlled crosses.

The invention, more specifically, provides a method for planttransformation comprising pollination pathway and silicon fibertreatment such that the delivery of transforming DNA into pollen grains.The invention provides a novel and non-obvious process that allows highfrequency of maize transformation, and in other crops as well. Besidehigh efficiency and low cost, its most important advantages are highreproducibility, genotype independence, genetic stability of thetransformants, and technical simplicity. The invention further providesa method for combining the pollination pathway and the delivering of thetransforming DNA into pollen grains by silicon carbide fibers whichtakes advantage of the natural reproduction system resulting intransformed zygotes.

The invention provides transgenic plants of the above-described method.

The invention also provides a paste comprising mixing silicon carbidefibers, pollen germination medium and DNA molecules.

Further objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows plants with sexual reproduction.

FIG. 2 depicts the effect of kanamycin on chlorophyll development inmaize seedling.

FIG. 3 depicts the non-transformed (left) and (presumably) transformedfor R gene (right) maize plants.

FIG. 4 depicts the effect of kanamycin on chlorophyll development inmaize isolated leaves.

FIG. 5 describes the reaction of a (putative) double transformation onlocal herbicide application.

DETAILED DESCRIPTION OF THE INVENTION

For the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

Definitions: Unless otherwise noted, terms are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. It addition to the definitions of terms provided below,definitions of common terms in molecular biology may also be found inLewin, Genes V, Oxford University Press: New York, 1994.

Genotype—The genetic complement of an organism.

Heterologous DNA—DNA from a source different than that of the recipientcell.

Homologous DNA—DNA from the same source as that of the recipient cell.

Hybrid—Progeny resulting from a cross between parental lines.

Inbred Lines—Organisms that are genetically homogeneous (homozygous)resulting from many generations of self crossing.

Monocot—Plants having a single cotyledon (the first leaf of the embryoof seed plants); examples include cereals such as maize, rice, wheat,oats and barley.

Non-Embryogenic Callus—A type of callus composed of undifferentiated,often highly vacuolated cells which are unable to be induced to formembryos.

Phenotype—Traits exhibited by an organism resulting from the interactionof genotype and environment.

Protoplast—Plant cells exclusive of the cell walls.

Somatic Cells—Body cells of an organism, exclusive of germinal cells.

Transformation—Acquisition of new genetic coding sequences by theincorporation of added (exogenous) DNA.

Transgenic—Organisms (plants or animals) into which new DNA sequencesare integrated.

The various fields of application of the present invention include, butare not limited to: (1) monocotyledoneous plants, especially cerealcrops (e.g., maize), where conventional transformation methods encounterserious (and frequently non-overcome) difficulties; (2) any floweringplant species with a high number of seeds per fruit (to be more exact,per unit artificial pollination, e.g., melon, tomato). The second groupcould be any plant species, if even other transformation methods havebeen used for it but were found technically complex; (3) gemnospermplants (e.g., pines). In summary, these fields of application could bepresented in the following tree.

The present invention addresses one or more of the foregoing or othershortcomings in the prior art by providing methods and products for thegenetic transformation of any plant species with sexual reproductionbased on a pollination-fecundation process using silicon carbide fibers.The present invention thus relates generally to methods and productsbased on a pollination-fecundation process. As used herein, the termtransgenic plants is intended to refer to plants that have incorporatedexogenous genes or DNA sequences, including but not limited to genes orDNA sequences which are perhaps not normally present, genes not normallytranscribed and translated (“expressed”) in a given cell type, or anyother genes of DNA sequences which one desires to introduce into thenon-transformed plant, such as genes which may normally be present inthe non-transformed plant but which one desires to have alteredexpression.

Exemplary genes which may be introduced include, for example, DNAsequences or genes from another species, or even genes or sequenceswhich originate with or are present in the same species, but areincorporated into recipient cells by genetic engineering methods ratherthan classical reproduction or breeding techniques. However, the termexogenous, is also intended to refer to genes which are not normallypresent in the cell being transformed, or perhaps simply not present inthe form, structure, etc, as found in the transforming DNA segment orgene, or genes which are normally present yet which one desires, e.g.,to have over-expressed. Thus, the term “exogenous” gene or DNA isintended to refer to any gene or DNA segment that is introduced into arecipient cell, regardless of whether a similar gene may already bepresent in such a cell.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicideresistance, increased yields, insect and disease resistance, physicalappearance, food content and makeup, etc. For example, one may desire toincorporate one or more genes encoding herbicide resistance. A potentialinsect resistance gene which can be introduced includes the Bacillusthuringiensis crystal toxin gene, which may provide resistance to pestssuch as lepidopteran or coleopteran. Protease inhibitors may alsoprovide resistance. Moreover, the expression of juvenile hormoneesterase directed towards specific insect pests may also haveinsecticidal activity, or perhaps cause cessation of metamorphosis.

Genes encoding proteins characterized as having potential insecticidalactivity, such as the cowpea trypsin inhibitor (CpTI) may find use as arootworm deterrent; genes encoding avermectin may prove particularlyuseful as a corn rootworm deterrent. Furthermore, genes encoding lectinsmay, additionally or alternatively, confer insecticide properties (e.g.,barley, wheat germ agglutinin, rice lectins), while others may conferantifungal properties (e.g., UDA (stinging nettle lectin), hevein,chitinase). It is proposed that benefits may be realized in terms ofincreased resistance to cold temperatures through the introduction of an“antifreeze” protein such as that of the Winder Flounder.

Ultimately, the most desirable “traits” for introduction into a monocotgenome may be homologous genes or gene families which encode a desiredtrait (e.g., increased yield per acre) and which are introduced underthe control of novel promoters or enhancers, etch, or perhaps evenhomologous or tissue specific (e.g., root specific) promoters or controlelements.

Because neither genomic or cDNA clones contain transcription andtranslation signals necessary for expression once transferred andintegrated into a plant genome, they must, therefore, be engineered tocontain a plant expressible promoter, a translation initiation codon(ATG), and a plant functional poly (A) additin signal (AATAAA) 3′ of itstranslation termination codon. Unique restriction enzyme sites at the 5′and 3′ ends of the cassette are typically included to allow for easyinsertion into a re-existing construct, such as a plasmid or phage.

Any of a number of transcription initiation regions (i.e., promoters)that direct transcription in plant cells is suitable. The promoter canbe either constitutive or inducible. It can be of bacterial origin,viral origin, or eukaryotic origin, such as plant origin. Examples ofconstitutive plant promoters useful for expressing genes in plant cellsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, maize ubiquitin (Ubi-1) promoter, rice actin (Act) promoter,nopaline synthase promoter, and the octopine synthase promoter. Avariety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals also canbe used for expression of foreign genes in plant cells, includingpromoters regulated by heat (e.g., heat shock promoters); light (e.g.,pea rbcS-3A or maize rbcS promoters or chlorophyll a/b-binding proteinpromoter); phytohormones, such as abscisic acid; wounding (e.g., wunI);anaerobiosis (e.g., Adh); and chemicals such as methyl jasmonate,salicylic acid, or safeners. Well known cell-, tissue-, organ-, andother developmental stage-specific promoters also can be used.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the gene toprovide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

If the mRNA transcribed from the genes is to be efficiently processed,DNA sequences which direct polyadenylation of the RNA are also commonlyadded to the vector construct. Polyadenylation sequences include, butare not limited to, the Agrobacterium octopine synthase signal, and thenopaline synthase signal. Replication sequences, of bacterial or viralorigin, or generally also included to allow the cassette to be cloned ina bacterial or phage host.

Selectable marker genes can be incorporated into the present expressioncassettes and used to select for those cells or plants which have becometransformed. The marker gene employed may express resistance to anantibiotic, such as kanamycin, gentamycin, G418, hygromycin,streptomycin, spectinomycin, tetracyline, chloramphenicol, and the like.Other markers could be employed in addition to or in the alternative,such as, for example, a gene coding for herbicide tolerance such astolerance to glyphosphate, sulfonylurea, phosphinothricin, orbromoxynil. Additional means of selection could include resistance tomethotrexate, heavy metals, complementation providing prototrophy to anauxotrophic host, and the like.

The particular marker employed will be one which will allow for theselection of transformed cells as opposed to those cells which are nottransformed. Depending on the number of different host species one ormore markers can be employed, where different conditions of selectionwould be useful to select the different host, and would be known tothose of skill in the art. A screenable marker such as theβ-glucuronidase gene can be used in place of, or with, a selectablemarker. Cells transformed with this gene can be identified by theproduction of a blue product on treatment with5-bromo-4chloro-3indoyl-β-D-glucuronide (X-Gluc).

In developing the present expression construct, i.e., expressioncassette, the various components of the expression construct such as theDNA molecules, linkers, or fragments thereof will normally be insertedinto a convenient cloning vector, such as a plasmid or phage, which iscapable of replication in a bacterial host, such as E. coli. Numerouscloning vectors exist that have been described in the literature. Aftereach cloning, the cloning vector can be isolated and subjected tofurther manipulation, such as restriction, insertion of new fragments,ligation, deletion, resection, insertion, in vitro mutagenesis, additionof polylinker fragments, and the like, in order to provide a vectorwhich will meet a particular need.

The above detailed description should not be construed to unduly limitthe present invention as modification and variations in the embodimentsdiscussed herein can be made by those ordinary skill in the art withoutdeparting from the spirit or scope of the present invention. The scopeof the invention is not to be considered limited thereto.

Conventional methods of gene isolation, molecular cloning, vectorconstruction, silicon carbon fiber techniques, and plant pollinationtechniques, etc., are well known in the art. One skilled in the art canreadily reproduce the plasmids vectors described below without undueexperimentation employing these methods in conjunction with the cloninginformation provided hereto. The various DNA sequences, fragments, etc.,necessary for this purpose can be readily obtained as components ofcommercially available plasmids and their applications in various plantspecies, or otherwise well known in the art.

The references cited herein evidence the level of skill in the art towhich the present invention pertains. The contents of each of thesereferences, including the references cited herein incorporated byreference by their entirety.

The present invention discloses a method for genetic transformation ofany plant species with sexual reproduction based on apollination-fecundation process. In essence, the method comprises thefollowing steps in which;

-   -   (a) preparing silicon carbide fibers solution;    -   (b) preparing pollen germination medium;    -   (c) mixing the silicon carbide fibers with DNA and with the        germination medium;    -   (d) putting fresh pollen into the above mixture resulting in a        paste;    -   (e) vortexing the mixture for 30-60 seconds;    -   (f) applying the resulting paste for pollination;    -   (g) selecting the transformants.

EXAMPLE 1 Genetic Transformation in Maize

Fertile transgenic maize plants were obtained by introducing thebacterial nptII gene encoding kanamycin resistance into zygotes by thepollination-fecundation process. The genomic copy of the gene Sh hasbeen transferred as well and its inheritance was detected in the progenyof stable transformants.

Three different plasmids were used for gene transfer experiments: pCT2T3and pGV1501 which carry the NOS promoter expressing the nptII gene as aselectable marker; the third plasmid, pBR322::Sh, contained a clonedgenomic copy of Sh Maize stocks used in the experiments were MK159, C22,W64B, Rad391139 and a multimarker line (ws31g1g12v4; wx sh). DNA wasapplied to silks of recipient plants as follows: a certain amount offresh pollen of a recipient plant, taken in time of full flowering, wasfirst immersed in the DNA solution. Then, immediately the paste-likepollen/DNA solution was placed onto the silks of the same recipientplant, thus producing self-pollination. Experimental plants yieldednearly 200 ears with about 25,000 seeds. The first stage of screeningfor transformants has been done using kanamycin resistance of seedlings.The selected seedlings were green and more vigorous than others, with amore developed root system. Kanamycin sensitive seedlings lostchlorophyll after 10-14 days (FIG. 2, see variant 2 and segregants invariant 3), discontinued their growth and eventually died.

The total DNA from selected resistant genotypes was analyzed usingSouthern-blot hybridization. The presence of hybridization zones of thenptII gene in the DNA of selected resistant seedlings was demonstrated.The hybridization test with DNA of control plants showed no positiveresults. Pollen from the above transformants was germinated on standardartificial media with kanamycin addition of 200 tg/ml. A ratio ofapproximately 1:1 of germinated to ungerminated pollen grains has beenobtained in this test. Pollen from control plants have not germinated atthe kanamycin concentration mentioned. The selfed progeny of fertiletransformants was evaluated in vitro using MSmedia with kanamycin.Segregation ratios of green to light-yellow (i.e., resistant tosensitive) close to 3.1 have been obtained. Southern-blot analysisshowed the presence of DNA sequences homologous to the nptII gene in thegenome of green genotypes and its absence in light-yellow ones.

A clone of the normal allele of the sh (shrunken) gene was also used asa selectable marker to overcome the problems associated with the invitro cultivation and to make the procedure of transformant selection assimple as possible. A multimarker line, ws31g1g12v4; wx sh, was used asa recipient in pollination experiments with pBR322::Sh plasmidcontaining a genomic copy of Sh gene in this case. As a result, earshave been found carrying some smooth seeds (presumed transformants Sh)the remaining seeds being shrunken (sh). No such exceptions have beenobserved in the control material.

The range of transformation frequency was 0.25-0.53, average 0.35. Aseries of tests have been conducted for evaluate the effect oftransformation on plant fertility. On the average, about 10-20% of theputative transformants have shown different morphological anomalies,including 5-10% of sterile plants. Another important question was thestability of the obtained transformants. All of the selectedtransformants for kanamycin appeared to segregate in the progeny; about10 homozygous lines were selected that were tested for stability tillT5, and two lines were tested till T8. Besides one case (out of ten),the material showed stable manifestation of resistance. The situationwith the Sh marker was quite different. In many cases (more than athird) the selected smooth seeds resulted in a selfed progeny withtotally mutant (not transformed) seeds. In other cases (less than 100/˜)the selfed progeny gave segregation ratios of the Sh sh closer to 13 (oreven less) than to the expected ratio 3:1.

Thus, genetic transformation in maize has been demonstratedsuccessfully, using different recipient lines and plasmids withdifferent markers by exploiting pollination-fecundation pathway todeliver alien genetic material into the embryo sac.

EXAMPLE 2 Silicon Carbide Fiber-Mediated Genetic Transformation in Maize

Maize transformation via the pollination pathway was initiated in orderto improve the pollination-based transformation technique. Severalplasmids with different selectable markers were used in pollinationexperiments of 600 maize plants of three different lines. We used thefollowing genes as selectable markers:

(i) neomycin phosphotransferase (npt II) gene encoding resistance to theantibiotic kanamycin (in the plasmid pBI121 which also contains the GUSreporter gene);

(ii) phosphinothricin acetyltransferase gene (bar) providing resistanceto the herbicide bialaphos in the plasmid pBARGUS. The advantage of thisconstruct is that it contains the Adh1 gene intron of maize which wasshown to increase the expression in maize; and

(iii) anthocyanin regulators (Cl) from maize controlling anthocyaninproduction (pAL69).

The study was performed using two maize lines: A619 and T403. We havetested the methods of DNA application using DNA concentrations 50-200ng/ml). The plasmids containing the different selectable markers usedfor this pollination experiment were used. Altogether 600 maize plantswere pollinated and 5,000 seeds were harvested.

These experiments showed promising results which support the proposedstrategy of maize transformation. Using silicon-fiber mediatedtransformation via the pollination pathway, we were able to producedozens of putative transformants. These plants showed expression ofanthocyanin caused by the transforming gene R and/or resistance to theherbicide ‘basta’ encoded by the Bar gene (FIG. 3) and kanamycinresistance (FIG. 4). An interesting and important result was thedemonstration of co-transformation in transposition. Using a mixture oftwo plasmids carrying the R gene and Bar gene we obtained putativetransformants for both. Moreover, it appeared that some putativetransformants for Bar manifested a strong increase in expression ofR-dependent anthocyanin synthesis after application of the herbicide(FIG. 5). This result corroborates the known involvement of anthocyaninsynthesis in stress reactions in plants.

These results indicate that the silicon fiber technique increased theefficiency of transformation several folds: the new treatment hasproduced 1.7% putative transformant (anthocyanin-expressing) seeds,while the old treatment gave only 0.35%. The results obtained with theold method (no fiber treatment) are rather similar to those obtained inKishinev.

Preparation of silicon carbide fibers: 50 mg of fibers (0.1-20 μmaverage diameter and 1.0-250 μm length, e.g., produced by AdvancedComposite Materials Corps, 1525 S. Buncombe Rd., Greer, S.C. 29651) areautoclaved in 1.5 ml tubes. Then, 5% solution is prepared by adding 1 mlsterile water.

Preparation of pollen germination medium: The solution contains 15%sucrose, 0.018% H₃BO₃, 0.04% Ca(NO₃)₂.4H₂O, pH 5.6 This solution isautoclaved for 20 minutes.

Mixing fibers with DNA and germination medium: Plasmid DNA in Tris EDTAsolution (25-100 μg dissolved) is mixed with 40 μl of fiber solution(5%), vortexed for a few seconds and incubated for 5 minutes at roomtemperature. Then, 500 μl of pollen germination medium is added.

Pollen treatment: Fresh pollen (200 mg) is put into 500 μl of the abovemixture and vortexed for 30-60 seconds.

Application of the treated pollen: The resulting paste is appliedimmediately for pollination, 250 μl for the silks of each ear. The earsare then covered by paper bags.

Selection of the transformants: The selection is performed oil the basisof specific cloned selectable markers which have either phenotypicexpression (e.g., anthocyanin) or provide resistance to some drugs (e.g.antibiotics or herbicides).

Our results obtained for several putative transformants selected foranthocyanin expression provide further confirmation for the presence ofthe foreign transforming DNA encoding for herbicide resistance (Basta)in the genome of these T1 plants and its transmission to the nextgeneration (T2 plants) (Table 1). TABLE 1 Results of Progeny Testing OfSome Putative Transformants Obtained In Co-Transformation ExperimentsPhenotypes of Segregation # of Anthocyanin T1 Basta as Anthocyanin T2Basta genotypes P/G R/S (P:G) (R:S) 424 P R 13:5  4:7 116 P S 13:5  0:298 P R 10:5  5:1 111 P S 9:10 2:2 121 P 8:11 3:0 45 P S 7:11 0:4 392 C0:12  4:14 49 P light S 7:11 1:1 Control C S 0:9  0:7Notes:P and G—pink and green color, R and S—resistant and susceptible.

1. A method for genetic transformation of any cereal plant species withsexual reproduction based on a pollination-fecundation processcomprising, (a) preparing a silicon carbide fibers solution; (b)preparing a pollen germination medium; (c) preparing a DNA solution; (d)preparing a mixture by mixing said silicon carbide fibers solution andsaid pollen germination medium with said DNA solution; (e) adding freshpollens into said mixture to form a paste; (f) vortexing said paste fora time interval of 30-60 seconds; (g) applying said paste forpollination; and (h) selecting for transformants; with the proviso thatsaid cereal plant species is not maize.
 2. A method for genetictransformation of any cereal plant species with sexual reproductionbased on a pollination-fecundation process according to claim 1, whereinsaid silicon carbide fibers are approximately 0.1-20 μm average diameterand 1-250 μm length.
 3. A method for genetic transformation of anycereal plant species with sexual reproduction based on apollination-fecundation process according to claim 1, wherein thepreferred size of said silicon carbide fibers is 1-2 μm diameter and10-80 μm length.
 4. A method for genetic transformation of any cerealplant species with sexual reproduction based on apollination-fecundation process according to claim 1, wherein an aqueoussolution for silicon carbide fibers is prepared by adding sterile wateror solvent to said fibers.
 5. A method for genetic transformation of anycereal plant species with sexual reproduction based on apollination-fecundation process according to claim 4, wherein saidsolution is 5% to 25% aqueous solution.
 6. A method for genetictransformation of any cereal plant species with sexual reproductionbased on a pollination-fecundation process according to claim 1, whereinsaid pollen germination medium is a solution containing about 5%-15%sucrose, 0.01%-1.0% H₃BO₃, 0.01%-1.0% Ca(NO₃)₂4H₂O at pH 5.6.
 7. Amethod for genetic transformation of any cereal plant species withsexual reproduction based on a pollination-fecundation process accordingto claim 1, wherein said preferred pollen germination medium is asolution containing about 15% sucrose, 0.018% H₃BO₃, 0.04% Ca(NO₃)₂4H₂Oat pH 5.6.
 8. A method for genetic transformation of any cereal plantspecies with sexual reproduction based on a pollination-fecundationprocess according to claim 1, wherein said DNA is a plasmid DNA.
 9. Amethod for genetic transformation of any cereal plant species withsexual reproduction based on a pollination-fecundation process accordingto claim 8, wherein said plasmid DNA is dissolved in a TE solution. 10.A method for genetic transformation of any cereal plant species withsexual reproduction based on a pollination-fecundation process accordingto claim 1, wherein said DNA solution is further incubated at about20-25° C.
 11. A method for genetic transformation of any cereal plantspecies with sexual reproduction based on a pollination-fecundationprocess according to claim 1, wherein the selection of a transformate isperformed by specific cloned selectable markers having a phenotypicexpression or providing resistance to some drugs.
 12. A method forgenetic transformation according to claim 1, wherein the selection of atransformate is performed by a specific closed selectable marker havinga phenotypic expression is an anthocyanin regulator.
 13. A method forgenetic transformation according to claim 11, wherein said selectablemarkers providing resistance to some drugs are antibiotics orherbicides.
 14. A method for genetic transformation according to claim13, wherein said selectable markers providing resistance to antibioticsis neomycin phosphotransferase gene or kanamycin gene.
 15. A method forgenetic transformation according to claim 11, wherein said selectablemarkers providing resistance to herbicides is phosphinothricinacetyltransferase gene.
 16. A method for genetic transformation of anycereal plant species with sexual reproduction based on apollination-fecundation process according to claim 1, in any cerealplant species with sexual reproduction comprising rice, wheat, oats andbarley.
 17. A paste comprising a silicon carbide fiber, a pollengermination medium, and a purified and isolated DNA molecule.
 18. Apaste as recited in claim 17 wherein said silicon carbide fibers having1-2 μm average diameter and 10-80 μm length.
 19. A paste as recited inclaim 17 wherein said silicon carbide fibers is a 5% aqueous solution.20. A paste as recited in claim 17 wherein said pollen germinationmedium is a solution containing about 15% sucrose, 0.018% H₃BO₃, 0.04%Ca(NO₃)₂4H₂O at pH 5.6.
 21. A paste as recited in claim 17 wherein saidDNA is a plasmid DNA.